Xerostomia, or dry mouth, is a common and devastating side effect of radiation therapy commonly used to treat head and neck cancer patients. No treatment currently exists to restore salivary function after radiation, and the only available therapies are palliative. Tissue engineering strategies for salivary gland regeneration enable cell encapsulation and survival, but do not fully replicate the intricate 3D patterning observed during development. 3D gradients of heparan sulfate proteoglycans (HSPGs) and heparin-binding growth factors (HBGFs) establish these patterns during development, and facilitate proper embryological and adult tissue organization and repair. Growth factor gradients within hydrogel biomaterials remain a target of tissue engineering researchers, but are rarely produced in a manner that replicates the matrix-directed action of native tissue. The goal of the research proposed herein is to establish biologically-inspired 3D gradients of covalently-bound HSPG within biologically-derived hyaluronic acid-based (HA) hydrogels, employ these to create complementary gradients of relevant HBGFs, and recapitulate the development of salivary glands to promote organized tissue regeneration. Our hypothesis is that HSPG/HBGF gradients within HA hydrogels will enable controlled, asymmetric development of morphology and phenotype of encapsulated primary human salivary cells, supporting branching morphogenesis and duct formation. In Aim 1, gradients of covalently-bound perlecan domain 1 (PlnD1) from the HSPG perlecan will be generated within HA hydrogels using multichannel devices and automated syringe pumps. Hydrogel characterization includes determination of PlnD1 coupling efficiency and gradient profiles. Functional HBGF binding capacity of these PlnD1 gradients will be determined using labeled versions of HBGFs such as FGF-7 and FGF-10, which promote salivary cell branching morphogenesis. In Aim 2, we will evaluate the effects of several PlnD1 and HBGF gradient profiles on encapsulated human salivary acinar-derived cells (hSACs) in vitro. Cell phenotype, motility, and organization will be assessed throughout culture in response to HBGF gradients. In Aim 3, HBGF/PlnD1 gradient hydrogels with encapsulated hSACs will be implanted in a rat model of salivary parotid gland damage and compared to controls. The salivary tissue will be evaluated via a functional amylase assay and histological scoring over several weeks to assess phenotypic function, implant organization, integration into native tissue, and overall tissue morphology. This research will enhance our understanding of cell response to gradients of HSPGs and morphogens for salivary tissue regeneration. Additionally, the research will describe a platform that will be broadly applicable for gradient delivery of any HBGF in regenerative medicine applications.